0013-7227/98/$03.00/0
Endocrinology
Copyright © 1998 by The Endocrine Society
Vol. 139, No. 10
Printed in U.S.A.
Development of Adrenal Zonation in Fetal Rats Defined
by Expression of Aldosterone Synthase and
11b-Hydroxylase
CHERYL WOTUS, BRETT K. LEVAY-YOUNG, LISA M. ROGERS,
CELSO E. GOMEZ-SANCHEZ, AND WILLIAM C. ENGELAND
Department of Surgery (C.W., B.K.L.-Y., L.M.R.) and of Cell Biology and Neuroanatomy (W.C.E.),
University of Minnesota, Minneapolis, Minnesota 55455; Department of Medicine, Endocrine Section
(C.E.G.-S.), Harry S. Truman Memorial Veterans Hospital, University of Missouri, Columbia, Missouri
65201
ABSTRACT
The adult rat adrenal cortex is comprised of three concentric steroidogenic zones that are morphologically and functionally distinguishable: the zona glomerulosa, zona intermedia, and the zona fasciculata/reticularis. Expression of the zone-specific steroidogenic
enzymes, cytochrome P450 aldosterone synthase (P450aldo), and
P450 11b hydroxylase (P45011b), produced by the zona glomerulosa
and zona fasciculata/reticularis, respectively, can be used to define
the adrenal cortical cell phenotype of these two zones. In this study,
immunohistochemistry and in situ hybridization were used to determine the ontogeny of expression of P450aldo and P45011b to monitor
the pattern of development of the rat adrenal cortex. RIA was used
to measure adrenal content of aldosterone and corticosterone, the
resulting products of the two enzymatic pathways. Double immunofluorescent staining for both enzymes at gestational day 16 (E16)
showed P45011b protein expressed in cells distributed throughout
most of the adrenal intermixed with a separate, but smaller, population of cells expressing P450aldo protein. Whereas expression of
P45011b protein retained a similar pattern of distribution from E16
to adulthood (ignoring distribution of SA-1 positive, presumptive
T
HE ADULT RAT adrenal gland is comprised of two
tissues with distinct developmental origins. The adrenal medulla consists of neuroectodermally derived chromaffin cells that produce catecholamines and numerous neuropeptides. The medulla originates outside the adrenal
cortex as a blastema of cells; these cells invade the cortical
tissue, disperse, and slowly migrate inward to aggregate in
the center of the gland around the time of birth (1, 2). The
mesodermally derived adrenal cortex consists of steroidogenic tissue, which is further divided into three morphologically and functionally distinguishable zones. The outer zona
glomerulosa produces the mineralocorticoid, aldosterone,
whereas the inner zona fasciculata/reticularis is the primary
producer of the glucocorticoid, corticosterone. The zona intermedia is positioned between the zona glomerulosa and
zona fasciculata/reticularis and lacks the ability to produce
either mineralocorticoids or glucocorticoids (3, 4).
Received March 4, 1998.
Address all correspondence and requests for reprints to: W. C. Engeland, Ph.D., Box 120 UMHC, 516 Delaware Street S.E., Minneapolis,
Minnesota 55455.
* This work was supported in part by NIH Grant GM-50150 and by
NSF Grant IBN-9319097.
medullary cells), P450aldo protein changed its pattern of distribution
by E19, becoming localized in a discontinuous ring of cells adjacent to
the capsule. By postnatal day 1, P450aldo protein distribution was
similar to that observed in adult glands; P450aldo-positive cells
formed a continuous zone underlying the capsule. In situ hybridization showed that the pattern of P45011b messenger RNA expression
paralleled protein expression at all times, whereas P450aldo messenger RNA paralleled protein at E19 and after, but was undetectable
before E19. However, adrenal aldosterone and corticosterone, as measured by RIA, were detected by E16, supporting the functional capacity of both phenotypes for all ages studied. These data suggest that
the development of the adrenal zona glomerulosa occurs in two distinct phases; initial expression of the glomerulosa phenotype in scattered cells of the inner cortex before E17, followed by a change in
distribution to the outer cortex between E17 and E19. It is hypothesized that this change in distribution occurs via cell differentiation,
rather than cell migration, and that a possible regulator of these
events is the fetal renin-angiotensin system. (Endocrinology 139:
4397– 4403, 1998)
There are two predominant theories that could be invoked
to explain the growth and differentiation that occur within
the fetal adrenal cortex, eventually leading to adult zonation.
The zonation theory hypothesizes that each zone develops
independently of the other (5). The cell migration theory
hypothesizes that cells proliferate in the zona glomerulosa,
then migrate and differentiate centripetally until they reach
the zona reticularis where they ultimately die (6). A similar
theory has also been used to explain the regeneration of the
adrenal cortex after enucleation or transplantation (7), implying that regeneration recapitulates the ontogeny of the
gland. Although previous investigations have attempted to
describe the timing of fetal adrenal zonation, the lack of
specific cell markers to define cortical phenotype has left
questions unanswered.
Past studies have relied on cellular ultrastructure, such as
the type of mitochondrial cristae or number of lipid droplets,
to characterize cortical phenotype throughout development
(8 –11). Although this method may be accurate for the identification of mature cortical cells, it is not clear whether ultrastructural criteria can be used to define a developing cell
and its functional capacity. In contrast, monitoring the expression of the genes that encode zone-specific steroidogenic
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4398
FETAL ADRENAL DEVELOPMENT
P450c11 enzymes could be an effective method for defining
the appearance of functional phenotypes during adrenal organogenesis. In the adult gland, production of 11b-hydroxylase (P45011b) and aldosterone synthase (P450aldo) is specific for the zona fasciculata/reticularis and the zona
glomerulosa, respectively. Mellon et al. (12) examined the
expression of P45011b and P450aldo messenger RNA
(mRNA) in RNA prepared from whole rat fetuses, offering
an approximation of when adrenal cells express zone-specific
steroidogenic enzymes. However, no attempt was made to
examine the distribution of different cellular phenotypes and
thus, this study could not delineate the pattern or timing of
zonal development. Recently, Mitani (13) examined the expression of P450aldo and P45011b protein in the developing
rat adrenal at the cellular level by immunohistochemistry;
however, studies were presented only for rats from embryonic day 18 (E18) and older and did not asses the ability of
the identified cells to produce steroids.
The present study defines the timing and pattern of development of the zona glomerulosa and the zona fasciculata/
reticularis in the fetal rat adrenal using in situ hybridization
histochemistry and immunohistochemistry. P450aldo and
P45011b mRNA and protein were monitored to identify areas within developing adrenals expressing the glomerulosa
or fasciculata/reticularis cell phenotype. RIAs for aldosterone and corticosterone in adrenal homogenates were used
to confirm the functional capacity of the cells expressing
these enzymes.
Materials and Methods
Materials
The following supplies and chemicals were purchased: isopentane
and ProbeOn slides from Fisher Scientific (Pittsburgh, PA); Tissue-Tek
O.C.T. Compound from Miles, Inc. (Elkhart, IN); Triton X-100, proteasefree BSA, and sodium azide from Sigma Chemical Co. (St. Louis, MO);
normal donkey serum (NDS), fluorescein-labeled donkey antimouse
(FITC-labeled DAM) antibody and Cy3-labeled donkey antirabbit (Cy3labeled DAR) antibody from Jackson ImmunoResearch Laboratories,
Inc. (West Grove, PA); SlowFade Antifade Kit from Molecular Probes,
Inc. (Eugene, OR); Biomax film and NTB-2 emulsion from Eastman
Kodak (Rochester, NY); corticosterone RIA kits from ICN Biomedical
(Costa Mesa, CA); aldosterone RIA kits from Diagnostic Products (Los
Angeles, CA); BCA protein assay kit from Pierce (Rockford, IL).
Animals
Male and day 14 timed-pregnant Sprague-Dawley rats (Harlan
Sprague-Dawley, Inc., Indianapolis, IN) were housed under a 12-h light,
12-h dark cycle (lights on 0600 –1800 h) with food and water available
ad libitum. Animals were maintained and cared for in accordance with
the NIH Guide for the Care and Use of Laboratory Animals. Experimental procedures were approved by the University of Minnesota Animal Care Committee.
Protocols
Tissue preparation. Pregnant and male adult rats were killed by CO2
asphyxiation; fetuses were immediately excised and decapitated; day 1
(D1) newborn rats were decapitated within 1–2 h after separation from
their mothers. Eight to sixteen adrenals from day 16 (E16), 17 (E17), 18
(E18), 18.5 (E18.5), 19 (E19), and 20 (E20) fetuses, D1 newborns and adult
male rats were collected for histological analysis. Adrenal glands were
removed, cleaned of surrounding fat, then mounted in O.C.T. compound and frozen in isopentane cooled in liquid nitrogen. Tissue sec-
Endo • 1998
Vol 139 • No 10
tions (14 mm) were cut in a cryostat, thaw-mounted on ProbeOn slides,
and stored desiccated at 220 C.
Analytical methods
Immunohistofluorescence. The P450aldo polyclonal antibody was generated by immunizing New Zealand White rabbits with the peptide MAPKVRQNARGSLTMDVQQ, corresponding to amino acids 175–192 of the
P450aldo enzyme. The P45011b monoclonal antibody was generated in
Swiss-Webster mice immunized with the peptide KNVYRELAEGRQQSC, corresponding to amino acids 272–285 of the P45011b enzyme, and conjugated to chicken serum albumin. The spleens corresponding to the mice with the highest titer were fused to a SP-2 myeloma
cell line, and the clones were selected for their ability to bind immobilized sonicated zona glomerulosa mitochondria from rats on a low
sodium diet. Immunohistofluorescent staining for P450aldo and
P45011b was simultaneously performed on tissue sections that were
fixed with Zamboni’s fixative (0.2% picric acid, 2% paraformaldehyde
in 0.16 m phosphate buffer) for 10 min at room temperature then washed
with PBS (3 3 5 min). Sections were blocked with NDS [1:10 in PBS with
0.1% Triton X-100 (PBST)] for 30 min at room temperature and incubated
with primary antisera (rabbit anti-P450aldo, 1:100 in PBST and mouse
anti-P45011b, 1:125 in PBST) for 16 –18 h at 4 C. Sections were then rinsed
in PBST (3 3 10 min) and incubated with secondary antibody (Cy3labeled DAR and FITC-labeled DAM, 1:200 and 1:100, respectively, in
PBST) for 1 h at room temperature. To reduce nonspecific labeling,
secondary antibodies were incubated before use with 10% normal rat
serum and 5% NDS for 1 h at room temperature and then centrifuged
at 1200 3 g at 4 C for 30 min; the supernatant served as the secondary
antibody. Sections were then rinsed in PBST (3 3 10 min) and then
coverslipped with SlowFade. Control slides consisted of preabsorbing
the anti-P450aldo antibody with a truncated version of the immunizing
peptide, using a nonimmune rabbit serum in place of anti-P45011b and
omitting the primary antibodies from the staining process. Control slides
were negative for staining for all developmental ages studied (data not
shown).
In situ hybridization. The in situ hybridization technique was performed
as outlined previously (14). Slides were incubated at 42 C overnight with
5–10 ng of 35S-labeled probe per ml of hybridization solution. Sections
were washed 5 3 15 min in 1 3 SCC at 54 C, dehydrated, air-dried, and
exposed to Biomax film. Some sections were dipped in NTB-2 emulsion
and exposed for 6 –10 days. Dipped slides were developed, counterstained with bisbenzimide and dehydrated.
Oligonucleotide probes, synthesized by Keystone Labs (Menlo Park,
CA), were designed to detect P450aldo [nucleotides (nt) 918 –953] (15)
and P45011b (nt 814 – 849) (16) mRNA. The specificity of probes used to
detect steroidogenic enzyme mRNAs was confirmed by three methods.
First, it was demonstrated that the hybridization signal could be blocked
by addition of excess (1003) unlabeled probe to the hybridization solution; second, for each antisense probe, hybridization was not affected
by addition of unlabeled probe antisense to another steroidogenic enzyme mRNA; and third, use of labeled sense probes for each of the
steroidogenic enzyme mRNA resulted in no hybridization signal (data
not shown). For the E18 adrenal sections, in situ hybridization was
performed by a modification of the method above (Levay-Young and
Engeland, submitted). Briefly, the probe sequences listed above were
resynthesized (Life Technologies, Bethesda, MD) with a 39 extension
(-59-A10GGGCCCCGGG-39) designed to hybridize to a second “labeling” oligonucleotide (59-T30CCCGGGGCCC-39). Probe and labeling oligonucleotides were hybridized and then extended with [33P]-dATP
(Amersham, Chicago, IL) and exo-Klenow DNA polymerase (Promega,
Madison, WI). The partially double stranded probes generated in this
way had approximately 10-fold higher specific activity than probes used
above, but the hybridization specificity of the modified probes was
unchanged. In addition, the [33P]-labeled probes produce the same pattern of hybridization for P450aldo and P45011b mRNA in fetal and adult
adrenals as that observed using the [35S]-labeled probes (data not
shown).
Photomicroscopy. Optical images were collected using a monochrome
CCD camera (Cohu, San Diego, CA), captured with a Scion LG-3 frame
grabber and processed on a Macintosh IIcx computer using the public
FETAL ADRENAL DEVELOPMENT
domain NIH Image 1.6 program (by W. Rasband (NIH) and available
from the Internet by anonymous ftp from zippy.nimh.nih.gov). Images
were then pseudocolored and overlapped with Adobe Photoshop 3.0 or
4.0 software.
RIA. Adrenals were homogenized with 20% ethanol in PBS, then centrifuged at 1200 3 g at 4 C for 5 min. Supernatants were collected for
steroid analysis, and pellets were resuspended in 1 n NaOH for measurement of protein content. Adrenal corticosterone and aldosterone
content were measured by RIA kits. The intraassay and the interassay
CV for corticosterone was 7.6% and 13.3%, respectively; the intraassay
and the interassay CV for aldosterone was 15.9% and 21.8%, respectively. Cross-reactivity for the corticosterone RIA, as defined by the
manufacturer, was 0.34% for deoxycorticosterone and 0.03% for aldosterone. Cross-reactivity for the aldosterone kit, as defined by the manufacturer and verified in our laboratory, was 0.033% for 18-OH-corticosterone and 0.002% for corticosterone. Steroid content was expressed
relative to adrenal protein measured using the BCA protein assay kit.
Statistical analysis. Values are expressed as the mean 6 sem; the range
is given where n 5 2. Before analysis, adrenal protein, aldosterone and
corticosterone content data were subjected to logarithmic transformation to reduce statistical variance. Differences between groups were
assessed by ANOVA. When ANOVA showed a significant difference
within an experiment, the Fisher’s LSD test was used to identify differences between groups. For all statistical analyses, differences were
considered significant when the test yielded a P , 0.05.
Results
Expression of P450aldo and P45011b protein
Double immunofluorescent staining was used to monitor
the time of appearance and spatial distribution of P45011b
and P450aldo positive cells in the fetal rat adrenal. At E16, the
earliest time examined, cells staining positively for P45011b
were detected throughout the adrenal (Fig. 1A). Nonstaining
areas correspond to medullary cells (defined as SA-1 positive
(17)/P45011b negative cells; data not shown) and blood vessels (11). The staining pattern for P45011b remained relatively constant from E16 to D1 (Fig. 1, A–E) as SA-1 positive/
P45011b negative medullary cells merged in the center of the
gland. Positive staining for P450aldo was also detected at
E16; however, staining was observed as single cells, or small
clusters of cells, dispersed throughout the adrenal (Fig. 1A).
This spatial distribution was markedly different from that
found in the adult gland, in which P450aldo staining was
limited to a 4- to 5-cell-wide zone under the capsule (Fig. 1F).
At E17, there appeared to be a decrease in the number of
P450aldo positive cells compared with the E16 gland (Fig.
1B), but the distribution was unchanged. By E18 and E18.5
an increased number of P450aldo positive cells was observed, and the distribution of these cells had changed from
E16, becoming more localized under the capsule and somewhat separate from the region of P45011b staining (Fig. 1C).
This is more clearly seen in E19 glands that demonstrated a
marked increase in P450aldo staining, occurring as a discontinuous ring of P450aldo positive cells adjacent to the
capsule with some residual staining within the region of
P45011b staining (Fig. 1D). At E19, a clear distinction between an outer and inner cortex was apparent; in addition,
a region of cells located between P450aldo and P45011b
positive cells did not stain, corresponding in enzymatic expression to the zona intermedia of the adult gland. Unlike the
scattered P45011b negative medullary cells seen at days 16 –
18.5, these zona intermedia-like P45011b negative cells were
4399
also SA-1 negative (data not shown). The postnatal day 1 (D1)
adrenals had a pattern of P450aldo staining that was similar
to that of the adult gland. (Fig. 1E). As in the adult, there were
no double labeled cells (i.e. cells positive for both P45011b
and P450aldo) detected in any fetal rat adrenal examined.
Expression of P450aldo and P45011b mRNA
In situ hybridization was used to determine the pattern of
P45011b and P450aldo mRNA expression at the same time
points in adrenal development as examined above. Expression of P45011b mRNA was similar to the pattern of P45011b
protein expression, being detected throughout the cortex in
all fetal, newborn (Fig. 2A, C, E, and G) and adult glands
examined and interspersed with P45011b negative patches of
presumptive medullary cells.
Expression of P450aldo mRNA was not detected in E16
and E17 glands (Fig. 2B). The absence of hybridization at
these time points is likely due to a lack of sensitivity of the
in situ hybridization technique used, combined with a low
level, single cell distribution of P450aldo expression, as was
seen at the protein level. Expression of P450aldo mRNA was
first detected in E18 adrenals as small, localized patches of
specific hybridization under the capsule (Fig. 2D). At E19,
P450aldo mRNA hybridization was in the form of a ring,
with intermittent unhybridized gaps, underlying the capsule
(Fig. 2F). This ring of P450aldo mRNA hybridization was
more continuous in D1 and adult glands (Fig. 2H), and
closely matched the distribution of P450aldo protein discussed above.
Measurement of aldosterone and corticosterone
Table 1 shows corticosterone and aldosterone content of
adult, D1 and fetal adrenals. Corticosterone was present in
all fetal adrenal homogenates and increased significantly
between E17 and E18. Corticosterone content then decreased
at E19 to markedly low levels at D1. Although the corticosterone content of adult adrenals was also lower than that
measured in all except E16 fetal glands, it was significantly
higher than the corticosterone content of D1 adrenals. Aldosterone was also present in all fetal adrenal homogenates
measured and increased between E17 and E18, and again
between E19 and D1. Unlike corticosterone content, there
was no difference in aldosterone content between D1 and
adult glands.
Discussion
Numerous attempts have been made to describe the ontogeny of adrenocortical zonation at the cellular level (8 –11).
The present study used in situ hybridization and immunohistochemistry to examine the expression of zone specific
P450 steroidogenic enzyme mRNAs and proteins, coupled
with steroid RIAs to examine cellular function. We thereby
describe the appearance of functional cortical cell phenotypes in the adrenal during development. In a recent report,
Mitani and colleagues (13) described the appearance of the
zona glomerulosa in the E20 adrenal gland based on staining
for P450aldo protein; staining of E18 and E19 adrenals failed
to demonstrate any expression. In contrast, the present study
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FETAL ADRENAL DEVELOPMENT
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Vol 139 • No 10
FIG. 1. Pseudocolored images of P450aldo (red) and P45011b (green) protein after double immunohistochemical staining in adrenals from E16
(A), E17 (B), E18.5 (C), E19 (D), D1 (E), and adult (F) rats. Cells staining positively for P450aldo are found scattered among cells staining for
P45011b in the inner cortex of E16 and E17 glands (A, with inset at higher magnification, and B). In the E18.5 gland (C) cells staining for
P450aldo are found in the outer cortex; note cell outside of the zone of P45011b staining (arrowhead). In E19 and older glands (D–F), cells staining
for P450aldo are localized in a subcapsular zone and separated from P45011b staining by a nonstaining intermediate zone. Nonstaining areas
in the inner cortex correspond to migrating medullary cells and blood vessels. ZG, Zona glomerulosa; ZF, zona fasciculata/reticularis; ZI, zona
intermedia. Bar, 100 mm.
FETAL ADRENAL DEVELOPMENT
4401
FIG. 2. Darkfield images of P45011b (left
panel) and P450aldo (right panel) mRNA
after in situ hybridization and emulsion
autoradiography (silver grains) in adrenals from E17 (A and B), E18 (C and D),
E19 (E and F) and D1 (G and H) rats.
Labeling of P45011b is homogeneous in
the inner cortex of fetal glands (A, C and
E); nonlabeled areas in the inner cortex
correspond to migrating medullary cells
or blood vessels. Background labeling is
seen in the E17 gland probed for
P450aldo (B). Patches of specific
P450aldo labeling underlie the capsule in
E18 glands (arrows, D). E19 and D1
glands exhibit a highly continuous ring of
P450aldo labeling under the capsule (F
and H). The adrenal capsule is delineated
by arrowheads. Bar, 100 mm.
clearly shows both P450aldo mRNA and protein expression
in E18 and E19 adrenals; the discrepancy in the observed
P450aldo expression at these time points may result from
differences in the reagents or detection systems used for
immunohistochemistry. The present study has also shown
that both glomerulosa and fasciculata/reticularis cell phenotypes are present in the adrenal as early as E16 and, as
demonstrated by RIA, are functionally competent in that
they produce aldosterone and corticosterone, respectively.
We believe that the lack of P450aldo mRNA visualization in
E16 and E17 glands is a reflection of levels of P450aldo
mRNA below the sensitivity of our assay, since immunohistochemical and RIA data convincingly support the presence of small numbers of glomerulosa cells in adrenals at
these ages.
The presence and distribution of fasciculata/reticularis
cells throughout the adrenal at E16, gradually changing to
produce an adult-like cortex surrounding a medulla at D1 is
not surprising. Corticosterone content of the developing adrenal increases between E17 and E18, and then drops sig-
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TABLE 1. Adrenal corticosterone and aldosterone content in fetal and newborn adrenals
E16
E17
E18
E19
E20
D1
Adult
Crown-rump length (cm)
Protein/adrenal (mg)
Corticosterone
(ng/mg protein)
Aldosterone
(ng/mg protein)
1.68 6 0.025 (4)
2.15 6 0.014 (17)a
2.63 6 0.027 (13)a
3.25 6 0.023 (19)a
3.37 6 0.024 (16)a
—
—
0.003; rng 5 0.018
0.014 6 0.001 (10)a
0.020 6 0.001 (10)a
0.030 6 0.002 (10)a
0.043 6 0.002 (10)a
0.037 6 0.006 (3)a
2.023 6 0.133 (4)a
997; rng 5 628
2003 6 126 (10)a
3382 6 352 (10)a
2636 6 187 (10)a
2205 6 138 (10)
16.0 6 2.21 (3)a
859 6 111 (4)a
2.04; rng 5 1.95
2.20 6 0.14 (10)
3.05 6 0.21 (10)a
2.93 6 0.15 (10)
4.33 6 0.27 (10)a
9.01 6 1.21 (3)a
9.62 6 2.00 (4)
For E16 –E20, 3–5 adrenals were pooled and homogenized for each protein and steroid sample. For D1 and adult measurements, 1 adrenal
was homogenized for each protein and steroid sample. Values represent mean 6 SEM for (n) samples; rng represents the range where n 5 2.
a
P , 0.05 vs. previous developmental age.
nificantly at birth, which is in agreement with previous studies (18 –20). Adrenal corticosterone content at D1 was
reduced relative to that measured in adult glands that had
been harvested after stress; this observation suggests that
reduced corticosterone content at D1 may reflect the onset of
the stress hyporesponsive period (21). The most novel finding of this study is that the ontogeny of the zona glomerulosa
appears to proceed through two developmental stages. The
first stage occurs as early as E16 and is characterized by the
presence of single or small clusters of glomerulosa cells distributed throughout the adrenal. During an interval between
E16 and E17, expression of the glomerulosa phenotype decreases in the inner gland, with only a few aldosterone producing cells remaining in the inner cortex. The second stage
is characterized by the appearance of functional glomerulosa
cells underlying the capsule between E18 and E19 and a
subsequent increase in cell number to produce an adult-like
zona glomerulosa by D1.
It is possible that the transition between the two stages of
zona glomerulosa development occurs via migration of glomerulosa cells from the inner to outer cortex. However, both
the rapid change in glomerulosa cell distribution, and the
virtual disappearance and subsequent reappearance of glomerulosa cells over a 2-day period, suggests that migration
is an unlikely mechanism. It seems more likely that glomerulosa cells present in the inner cortex at E16 either differentiate
into fasciculata/reticularis cells (22) or die (23) between E16
and E17. Subsequently, cells adjacent to the capsule would
differentiate to become glomerulosa cells and then proliferate (24) to form the adult-like zonation. Based on work in
adult adrenal regeneration and plasticity, potential progenitors of the glomerulosa phenotype include capsular (7, 25,
26) and intermediate (3) cells. The early expression of
P45011b denoting the zona fasciculata/reticularis, followed
by the appearance of P450aldo in cells underlying the capsule
in late gestation, supports the independent establishment of
each zone during fetal development as proposed by the
zonation theory (5).
The mechanisms that mediate the two stages of zona glomerulosa development are unknown; we favor the hypothesis that each stage is regulated independently of the other.
The disappearance of P450aldo positive cells between E16
and E17 following a presumed differentiation from an undifferentiated precursor cell is reminiscent of transient 17ahydroxylase mRNA expression in a subpopulation of mouse
adrenal cells between E12.5 and birth (27), and of P45011B3
gene expression that is elevated only in neonatal adrenals (28,
29). The subsequent abrupt appearance of P450aldo protein
and mRNA in the adult-like subcapsular zona glomerulosa
toward the end of gestation suggests that classic regulators
of glomerulosa function initiate this event, independent of
the earlier events.
One potential regulator of this second stage of zona glomerulosa development is the renin-angiotensin system. It
has been shown that midgestational fetal bovine cells respond to AII through the AII type 1 receptor (AT1) by increasing P450c18 activity (the aldosterone synthase P450 enzyme found in bovine adrenals) and aldosterone production
in vitro (30). In the rat, transcripts for both AT1 receptor
subtypes (AT1A and AT1B) are found in the zona glomerulosa
beginning at E17, and AT1A mRNA expression peaks at E19
(31). The AII type 2 receptor (AT2) has also been identified
in the periphery of the developing adrenal by E15 (32). Because the AT1 receptor is implicated as the primary receptor
through which AII regulates aldosterone production, and the
AT2 receptor may also regulate growth and differentiation in
developing tissues, it follows logically that AII may upregulate expression of P450aldo in the outer cortex during
late gestation. Of course, this raises the question of what
regulates AII receptor subtype expression in the zona
glomerulosa.
Whereas the relevance of glucocorticoids in the normal
development of the fetus is well documented (33), a similar
role for mineralocorticoids is still unknown. None the less,
it is likely that fetal aldosterone production has physiological significance during development. Recently, Brown
et al. (34) investigated the ontogeny of mineralocorticoid
receptor (MR) mRNA and 11b-hydroxysteroid dehydrogenase type 2 (11b-HSD2) mRNA expression in the mouse
and found co-expression of these mRNAs in the kidney
and colon between E17.5 and birth. Because 11b-HSD2 is
believed to inactivate glucocorticoids, which have a higher
affinity for the MR than aldosterone, these data support
the idea that aldosterone produced late in gestation can
bind to MR in fetal tissues to affect growth or function.
Although it is not clear what function glomerulosa cells
present in the gland before E19 have in fetal physiology,
it will be interesting to determine what controls the initial
appearance and final fate of these early P450aldo expressing cells. These issues will be clarified by a careful examination of the differentiation of adrenal steroidogenic cells
during development.
FETAL ADRENAL DEVELOPMENT
Acknowledgments
We thank Debra Fitzgerald for technical assistance in the RIA of
adrenal steroids.
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